High-throughput application of CRISPR technology to identify gene function in Salmonella

高通量应用CRISPR技术鉴定沙门氏菌基因功能

• 批准号: 9172073
• 负责人: Joseph Thomas Wade
• 金额: $ 20.96万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-25 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9172073
• 关键词: Address; BCAR1 gene; Bacteria; Bacterial Genes; Bacterial Genome; Bacterial Physiology; CRISPR interference; CRISPR/Cas technology; Cessation of life; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Effectiveness; Engineering; Escherichia coli; Essential Genes; Future; Gene Deletion; Genes; Genetic Transcription; Genomic approach; Genomics; Growth; Homologous Gene; Knowledge; Methodology; Methods; Phenotype; Positioning Attribute; Salmonella; Salmonella enterica; Study models; System; Technology; Work; Yeasts; base; deletion library; gene function; gene repression; genome-wide; innovation; next generation sequencing; pathogen; promoter; tool

SUMMARY Our understanding of bacterial physiology is limited by the fact that the majority of bacterial genes are uncharacterized. Even in the best studied model bacterium, Escherichia coli K-12, >1,200 genes are completely uncharacterized, and for many of the “characterized” genes, very little functional information is known. Our ability to characterize gene functions is now outpaced by identification of new genes with unknown function. Hence, it is critical that we develop high-throughput methodologies to reliably assign gene function at a more rapid pace. Two such high-throughput methods, Chemical Genomics and SGA, have been developed for use in yeast, and subsequently applied in E. coli. However, these methods are labor-intensive, provide little information about essential genes, and are only readily applicable in species with one (for chemical genomics) or two (for SGA) deletion collections (deletion collections are available for only a few bacterial species). Furthermore, SGA is only applicable to species in which pairs of gene deletions can be easily combined. We will harness the combined power of CRISPR interference (CRISPRi) and next-generation sequencing technologies to redesign the Chemical Genomics and SGA approaches. Our preliminary data demonstrate the effectiveness of this approach in E. coli and establish CRISPRi in Salmonella. CRISPRi-based methods for Chemical Genomics and SGA have three major advantages over the established approaches. First, CRISPRi- based methods are far less labor intensive and can be applied more rapidly and more cheaply than the existing approaches. Second, CRISPRi-based methods are more effective for studying essential genes. Third, CRISPRi- based methods are readily applicable to a wide range of bacterial species, including species that lack deletion collections. We expect to identify groups of functionally related genes using each approach individually, and by combining data from both approaches. We expect that the relationships we identify between genes will be the basis of many future studies, as has been the case for Chemical Genomics and SGA data in yeast and E. coli. Moreover, our work will have a long-term impact by establishing these methods as facile, high-throughput tools for investigating gene function in a wide variety of bacterial species. The proposed work is highly significant because it provides a powerful solution to the major problem of identifying gene function. The proposed work is innovative because no prior studies have applied CRISPRi to Chemical Genomics or SGA. Moreover, there have been no previous high-throughput studies of gene function in Salmonella.

概括 我们对细菌生理学的理解受到大多数细菌基因的事实的限制 未表征。即使在最好的研究菌模型细菌中，大肠杆菌K-12，> 1200个基因也完全 未表征，对于许多“特征”基因，知名度很少。我们的能力 现在，通过识别具有未知功能的新基因来表征基因函数。因此，它 至关重要的是，我们开发高通量方法以更快的速度分配基因函数。 已经开发了两种这种高通量方法，即化学基因组学和SGA，用于酵母，以及 随后应用于大肠杆菌。但是，这些方法是劳动密集型的，很少提供有关 必需基因，仅适用于具有（化学基因组学）或两种物种（用于SGA）的物种 缺失收集（仅适用于几种细菌物种的缺失收集）。此外，SGA只是 适用于很容易组合基因缺失对的物种。 我们将利用CRISPR干扰（CRISPRI）和下一代测序的综合力量 重新设计化学基因组学和SGA方法的技术。我们的初步数据证明了 这种方法在大肠杆菌中的有效性并在沙门氏菌中建立CRISPRI。基于CRISPRI的方法 化学基因组学和SGA在既定方法中具有三个主要优势。首先，crispri- 基于基于的方法要少得多，并且可以比现有的更迅速，更便宜地应用 方法。其次，基于CRISPRI的方法更有效地研究基本基因。第三，crispri- 基于的方法很容易适用于多种细菌，包括缺乏缺失的物种 收藏。 我们希望使用每种方法单独识别与功能相关的基因组，并结合 来自两种方法的数据。我们希望我们在基因之间识别的关系将是许多人的基础 未来的研究，就像酵母和大肠杆菌中化学基因组学和SGA数据一样。而且，我们的 工作将通过建立这些方法作为调查的轻松，高通量工具，从而产生长期影响 基因在多种细菌中的功能。拟议的工作非常重要，因为它提供了 识别基因功能的主要问题的有力解决方案。拟议的工作是创新的，因为 没有先前的研究将CRISPRI应用于化学基因组学或SGA。而且，没有以前 沙门氏菌中基因功能的高通量研究。